High-quality bacterial genomes without the complexity
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- Published on: February 5 2026
In the past, bacterial genome assembly has relied on short-read sequencing, which struggles to resolve repetitive regions longer than the library insert size1. Oxford Nanopore sequencing offers a different approach. By generating reads of any length, it overcomes this problem and offers a simple and cost-effective method for generating reference-quality bacterial genome assemblies.
Researchers are now showing that near-complete bacterial genomes can be assembled using nanopore sequencing alone, without costly short-read polishing or complex data analysis pipelines. In parallel, other teams are using these high-quality assemblies to reveal insights about a pathogen notoriously challenging to analyse.
Simplifying bacterial genome assembly from complex samples
Improvements in Oxford Nanopore sequencing accuracy, following the release of Kit 14 chemistry, prompted Professor Mads Albertson at Aalborg University, Denmark, to ask whether short-read polishing of bacterial genome assemblies is still necessary1.
To find out, Sereika and Kirkegaard et al. sequenced bacterial samples from a mock community using our technology1. At 40x coverage, they assembled near-finished bacterial genomes without any polishing (Figure 1). From here, the team assessed performance on metagenomic genome assembly by sequencing a sample of activated sludge, and they reached the same conclusion: nanopore-only data generated near-complete microbial genomes without short-read polishing1.
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Figure 1. Indels observed per 100 kb in the de novo bacterial isolate assemblies at different depths of coverage, both with and without short-read polishing. The authors noted that short-read polishing of nanopore data obtained using Kit 14 chemistry with R10.4 Flow Cells provided no significant improvement in assembly quality. Image adapted from Sereika and Kirkegaard et al.1 and available under Creative Commons license (creativecommons.org/licenses/by/4.0).
'Oxford Nanopore R10.4 enables the generation of near-finished microbial genomes from pure cultures or metagenomes at coverages of 40-fold without short-read polishing'
Sereika, M. and Kirkegaard,R.H. et al.1
In a subsequent study, the team demonstrated that Oxford Nanopore sequencing generates high-quality bacterial genomes from complex terrestrial samples. Sequencing 154 complex soil and sediment samples, Sereika et al.2 recovered over 15,000 metagenome-assembled genomes (MAGs) using nanopore sequencing alone. They identified thousands of ‘previously undescribed microbial species’ without enrichment or culture2.
Other researchers have used similar culture-independent sequencing methods to explore hidden microbial communities in soil microbiomes. For example, Burian et al. generated complete and near-complete MAGs from soil samples, revealing previously undiscovered bioactive molecules that could form the basis of new antibiotics3.
While these studies highlight the power of nanopore sequencing for cost-effective, high-quality microbial genome assembly, some bacterial pathogens remain particularly challenging to analyse; most notably, Mycobacterium tuberculosis4.
Why M. tuberculosis is challenging to analyse
M. tuberculosis (TB) is the pathogen responsible for tuberculosis, which remains one of the deadliest infectious diseases, causing 10.7 million new cases in 2024 and resulting in 1.23 million deaths that year5. Plus, drug-resistant TB is a particularly significant threat to public health1,4. Due to this, genome sequencing has become central to TB research and surveillance, including for strain tracking, transmission analysis, and the identification of drug-resistance mutations4.
Short-read sequencing technology has typically been relied on to investigate the genetic basis of resistance and the genomics underpinning TB transmission. However, it struggles with the high-GC content and repetitive regions of the TB genome, particularly the GC-rich pe/ppe genes associated with drug resistance, which are then often excluded from analysis. Also, the high capital cost and centralisation associated with these sequencing platforms limit access to genomic analysis in many areas with a high TB burden and low income4,6.
Accessing the entire TB genome
In contrast, our platform produces sequencing reads of unrestricted length. It has been recognised as a 'promising platform for cost-effective application' to TB genome analysis4.
Testing out the capabilities of our technology, Gómez-González et al.4 compared the performance of Oxford Nanopore sequencing and Illumina short-read sequencing for analysis of drug susceptibility prediction and outbreak investigation. When the researchers sequenced TB clinical research isolates, nanopore sequencing delivered improved coverage across repetitive regions, detecting variants more comprehensively than the Illumina platform4.
As expected from long reads, the team detected a higher number of large variants with nanopore reads than with Illumina reads. Despite this, all lineage predictions were identical between the two platforms (Figure 2), but only nanopore data successfully resolved the pe/ppe regions. This meant the researchers could incorporate single nucleotide variants (SNVs) for lineage analysis at these important genes, providing valuable data in ‘outbreak settings, where transmission analysis of closely related isolates can be potentially better established’4.
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Figure 2. Phylogenetic trees representing the branching order for the TB clinical research isolates studied, showing equal branch lengths for the 10 pairs of sequenced isolates. Figure from Gómez-González et al.4 and available under Creative Commons license (creativecommons.org/licenses/by/4.0).
Revealing understudied genomic variants in TB
In another study, Hall et al.6 aimed to establish whether nanopore data could be used to reproduce equivalent transmission clusters and drug susceptibility profiles to those generated with Illumina short-read data6. To investigate this, the team obtained matched nanopore and Illumina data from 151 isolates. They found that isolate clustering was the same between the two platforms, and in terms of genotyping resistance-associated SNVs and indels, the team obtained near-identical results, with a concordance of >99.99% between the two technologies6.
'Our analysis shows that it is now possible to obtain high-precision SNP calls in M. tuberculosis with current nanopore data'
Hall, M.B. et al.5
Going beyond concordance with short-read data, Canalda-Baltrons et al.7 demonstrated that nanopore sequencing reveals more genomic information about TB than other technologies. Using only our technology, the researchers identified structural variants (SVs) potentially associated with resistance to first- and second-line drugs, highlighting ‘the broader role of SVs in shaping [TB] diversity’, and their importance in tackling drug-resistant TB7.
Together, these studies show that Oxford Nanopore sequencing can deliver near-complete bacterial genomes from complex samples as a standalone platform. From environmental microbiomes to challenging pathogens such as TB, our technology opens new opportunities for microbial genomics and infectious disease research.
Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.
Sereika, M. and Kirkegaard, R.H. et al. Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing. Nat. Methods 19(7):823–826 (2022). DOI: https://doi.org/10.1038/s41592-022-01539-7
Sereika, M. et al. Genome-resolved long-read sequencing expands known microbial diversity across terrestrial habitats. Nat. Microbiol. 10(8):2018–2030 (2025). DOI: https://doi.org/10.1038/s41564-025-02062-z
Burian, J.B. et al. Bioactive molecules unearthed by terabase-scale long-read sequencing of a soil metagenome. Nat. Biotechnol. Online ahead of print (2025). DOI: https://doi.org/10.1038/s41587-025-02810-w
Gómez-González, P.J. et al. Portable sequencing of Mycobacterium tuberculosis for clinical and epidemiological applications. Brief. Bioinform. 23(5):bbac256 (2022). DOI: https://doi.org/10.1093/bib/bbac256
WHO. Tuberculosis. Available at: https://www.who.int/news-room/fact-sheets/detail/tuberculosis. [Accessed 4 February 2026]
Hall, M.B. et al. Evaluation of nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation: a genomic analysis. Lancet Microbe 4(2):e84–e92 (2023). DOI: https://doi.org/10.1016/s2666-5247(22)00301-9
Canalda-Baltrons, A. et al. Genome graphs reveal the importance of structural variation in Mycobacterium tuberculosis evolution and drug resistance. Nat. Commun. 16(1):10746 (2025). DOI: https://doi.org/10.1038/s41467-025-65779-9
